1. How do you identify and clone a gene of interest? Shotgun approach? Is there a better way?

2. DNA Libraries What is a DNA library? 2 main types Genomic Library cDNA Library

3. Genomic Library Random digestion of genome by restriction enzyme Fragments are cloned into a vector Recombinant vectors used to transform bacteria Disadvantages Non-coding regions (introns) of DNA Needle in a haystack

4. cDNA Library Double stranded DNA is made from mRNA Reverse Transcriptase DNA polymerase Linker sequences added Contain restriction sites Cloned into bacteria Advantages No introns, actively expressed genes Need abundant amount of mRNA

5. Library Screening Now that you have a library, how do you find the gene of interest? Colony Hybridization

6. Transformed bacteria are grown on an agar plate Cells are transferred to membrane Membrane is probed with radioactive oligonucleotide

7. Colony Hybridization Autoradiography will show which colonies contain the gene of interest The colony can be chosen and grown to isolate the plasmid

8. Cloning by PCR

9. What can you do with a cloned gene? Restriction Mapping DNA sequencing (we’ll cover later) Chromosomal location and copy number FISH Southern Blotting Study gene expression

10. Fluorescence in situ Hybridization

11. What can you do with a cloned gene? Restriction Mapping DNA sequencing (we’ll cover later) Chromosomal location and copy number FISH Southern Blotting Study gene expression

12. Studying Gene Expression Northern Blotting Reverse Transcription PCR (RT-PCR) Real-time PCR (qPCR) In situ hybridization DNA Microarray (we’ll discuss later) Protein expression and purification Gene mutagenesis studies RNA Interference (RNAi)

13. Northern Blotting Similar to Southern blot Use probes to detect RNA Can be used to study mRNA expression in different tissues

14. Reverse Transcription PCR Useful with small amounts of RNA RNA cannot be amplified by PCR Use reverse transcriptase to make double stranded cDNA Primers are used to amplify the cDNA Run on a gel Allows to see expression patterns

15. Real-time PCR Aka quantitative PCR (qPCR) Allows researcher to quantify amplification reactions in real time Requires expensive thermocycler Reaction tube contains dye that glows from a laser when bound to double stranded DNA SYBR Green or TaqMan probes No need to run a gel

16. Real-time PCR

17. in situ Hybridization Use fluorescent probes to locate gene of interest in a tissue

18. Protein Expression and Purification Use transformed bacteria to produce protein from gene of interest More later

19. Gene Mutagenesis Studies aka site directed mutagenesis Mutations created at specific nucleotides Express in cells to see what happens

20. RNA Interference (RNAi)

21. Applications of DNA Technology

22. The Human Genome Project Goals Analyze genetic variations among humans Map and sequence genomes of model organisms Develop new lab technologies Disseminate information about the genome Consider ethical, legal, and social issues of genetic research

23. The Human Genome Project